Monoclonal antibodies used in the diagnosis and early development of prostate cancer

Abstract

The present invention provides monoclonal antibodies designed for the diagnosis and early detection of prostate cancer, and in particular relates to monoclonal antibodies developed to target the specificity of the secreted soluble ADAM9 variant (sADAM9v2) in the vicinity of prostate cancer.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 377,801, filed September 30, 2022. The entirety of the aforementioned application is incorporated herein by reference.

[0002] Sequence Listing Description The contents of the electronic sequence listing named 23P0419_final.xml (size: 12,288 bytes, created date: September 26, 2023) are incorporated herein by reference in their entirety.

[0003] FIELD OF THE INVENTION The present disclosure relates to monoclonal antibodies designed for the diagnosis and early detection of prostate cancer, and in particular to monoclonal antibodies developed to target the specificity of soluble ADAM9 variants secreted in the vicinity of prostate cancer. [Background technology]

[0004] Background of the Invention Based on Taiwan's 2021 National Cancer Registry data, prostate cancer is the sixth leading cause of incidence and the fifth leading cause of mortality among Taiwanese men. Notably, early signs of prostate cancer are usually subtle and resemble those of prostate hyperplasia, leading to potential oversight. Prostate cancer only gains meaningful attention when it invades the seminal vesicles, presenting symptoms such as hematospermia or painful ejaculation, or metastasizes to other organs. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO 99 / 58572 [Patent Document 2] U.S. Patent No. 5,565,332 [Patent Document 3] U.S. Patent No. 5,580,717 [Patent Document 4] U.S. Patent No. 5,733,743 [Patent Document 5] U.S. Patent No. 6,265,150 [Patent Document 6] PCT Publication No. WO 87 / 04462 [Patent Document 7] U.S. Patent No. 4,485,045 [Patent Document 8] U.S. Patent No. 4,544,545 [Patent Document 9] U.S. Patent No. 5,013,556 [Patent Document 10] U.S. Patent No. 3,773,919 [Patent Document 11] PCT Publication No. WO 00 / 53211 [Patent Document 12] U.S. Patent No. 5,981,568 [Patent Document 13] PCT Publication No. WO 90 / 07936 [Patent Document 14] PCT Publication No. WO 94 / 03622 [Patent Document 15] PCT Publication No. WO 93 / 25698 [Patent Document 16] PCT Publication No. WO 93 / 25234 [Patent Document 17] PCT Publication No. WO 93 / 11230 [Patent Document 18] PCT Publication No. WO 93 / 10218 [Patent Document 19] PCT Publication No. WO 91 / 02805 [Patent Document 20] U.S. Patent No. 5,219,740 [Patent Document 21] U.S. Patent No. 4,777,127 [Patent Document 22] British Patent No. 2,200,651 [Patent Document 23] European Patent No. 0 345 242 [Patent Document 24] PCT Publication No. WO 94 / 12649 [Patent Document 25] PCT Publication No. WO 93 / 03769 [Patent Document 26] PCT Publication No. WO 93 / 19191 [Patent Document 27] PCT Publication No. WO 94 / 28938 [Patent Document 28] PCT Publication No. WO 95 / 11984 [Patent Document 29] PCT Publication No. WO 95 / 00655 [Patent Document 30] U.S. Patent No. 5,814,482 [Patent Document 31] PCT Publication No. WO 95 / 07994 [Patent Document 32] PCT Publication No. WO 96 / 17072 [Patent Document 33] PCT Publication No. WO 95 / 30763 [Patent Document 34] PCT Publication No. WO 97 / 42338 [Patent Document 35] PCT Publication No. WO 90 / 11092 [Patent Document 36] U.S. Patent No. 5,580,859 [Patent Document 37] U.S. Patent No. 5,422,120 [Patent Document 38] PCT Publication No. WO 95 / 13796 [Patent Document 39] PCT Publication No. WO 94 / 23697 [Patent Document 40] PCT Publication No. WO 91 / 14445 [Patent Document 41] European Patent No. 0524968 [Non-patent literature]

[0006] [Non-Patent Document 1] Kabat, EAら, (1991) Sequences of Proteins of Immunological Interest, 5th edition, US Ministry of Health and Welfare, NIH Publication No. 91-3242 [Non-licensed Document 2] Lefranc, M.-P., Nucleic Acids Res., 27:209-212 (1999) [Non-licensed Document 3] Ruiz, M., Nucleic Acids Res., 28:219-221 (2000) [Non-licensed Document 4] Lefranc, M.-P., Nucleic Acids Res., 29:207~209 pages (2001) [Non-licensed Document 5] Lefranc, M.-P, Nucleic Acids Res., 31:307~310 pages (2003) [Non-licensed Document 6] Lefranc, M.-P., In Silico Biol., 5, 0006 (2004) [Epub], pages 5:45~60 (2005) [Non-licensed Document 7] Lefranc, M.-P., Nucleic Acids Res., 33:D593~597 (2005) [Non-licensed Document 8] Lefranc, M.-P., Nucleic Acids Res., 37:D1006~1012 (2009) [Non-licensed Document 9] Lefranc, M.-P., Nucleic Acids Res., 43:D413~422 (2015) [Non-licensed Document 10] Chothiaら、(1989) Nature 342:877 [Non-licensed Document 11] Chothia. C. (1987) J. Mol. Biol. 196: 901-917 [Non-licensed Document 12] Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948 [Non-Patent Document 13] Almagro, J. Mol. Recognit. 17:132-143 (2004) [Non-Patent Document 14] Molecular Cloning: A Laboratory Manual, edited by J. Sambrook et al., 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 [Non-Patent Document 15] Current Protocols in Molecular Biology, edited by E. M. Ausubel et al., John Wiley & Sons, Inc., New York [Non-Patent Document 16] Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York [Non-Patent Document 17] Winters et al. (1994) Anno. Rev. Immunol. 12:433-455 [Non-Patent Document 18] McCafferty et al. (1990) Nature 348:552-553 [Non-Patent Document 19] Morrison et al. (1984) Proc. Nat. Acad. Sci. 81:6851 [Non-Patent Document 20] Harlow and Lane, Chapter 11 of Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999 [Non-Patent Document 21] Brown, M. et al., Cell, 49:603-612 (1987) [Non-licensed Document 22] Gossen, M. and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547~555115 (1992) [Non-licensed Document 23] Yao, F.ら, Human Gene Therapy, 9: pp. 1939~1950 (1998) [Non-licensed Document 24] Shockelt, P., Proc. Natl. Acad. Sci. USA, 92:6522~6526 pages (1995) [Non-licensed Document 25] M. Brown, Cell, 49:603~page 612 (1987) [Non-licensed Document 26] Gossen and Bujard (1992) [Non-licensed Document 27] M. Gossen, Natl. Acad. Sci. USA, 89:5547~5551 pages (1992) [Non-licensed Document 28] Yaoら、Human Gene Therapy [Non-licensed Document 29] Gossen 5ら, Natl. Acad. Sci. USA, 89:5547~5551 pages (1992) [Non-licensed Document 30] Shockett, Proc. Natl. Acad. Sci. USA, 92:6522~6526 pages (1995) [Non-licensed Document 31] Remington: The Science and Practice of Pharmacy, 20th Edition (2000) Lippincott Williams and Wilkins, KE Hoover, editors [Non-licensed Document 32] Skoog, DA; West, D M.; Holler, JF; Crouch, SR (2004). "Chapter 14~16" Fundamentals of Analytical Chemistry (8th Edition) [Non-licensed Document 33] Douglas W. Haywick, (2007~2008). "Elemental Chemistry" [Non-licensed Document 34] Remington: The Science and Practice of Pharmacy, 20th edition, (2000) Lippincott Williams and Wilkins, KE Hoover, editors [Non-licensed Document 35] Epstein, ら, Proc. Natl. Acad. Sci. USA 82:3688 (1985) [Non-licensed Document 36] Hwang, ら, Proc. Natl. Acad. Sci. USA 77:4030 (1980) [Non-licensed Document 37] Remington, The Science and Practice of Pharmacy, 20th Edition, Mack Publishing (2000) [Non-licensed Document 38] Findeis, Trends Biotechnol. (1993) 11:202 [Non-licensed Document 39] Chiou, Gene Therapeutics: Methods and Applications of Direct Gene Transfer (JA Wolff, ed.) (1994) [Non-licensed Document 40] Wuら, J. Biol. Chem. (1988) 263:621 [Non-licensed Document 41] Wuら, J. Biol. Chem. (1994) 269:542 [Non-licensed Document 42] Zenke, Proc. Natl. Acad. Sci. USA (1990) 87:3655 [Non-licensed Document 43] Wuら, J. Biol. Chem. (1991) 266:338

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[0007] Therefore, effective methods for early detection of prostate cancer are of paramount importance. [Means for solving the problem]

[0008] SUMMARY OF THE INVENTION ADAM9 has been shown to increase its expression in various cancer epithelial cells, such as prostate, breast, kidney, and lung cancer. Enhanced expression is positively correlated with cancer progression and metastasis tendency. Research has shown that the predominant protein expression may be due to soluble ADAM9 (sADAM9).

[0009] At the same time, two distinct splice variants of sADAM9 have been identified, including sADAM9v1 reported by Mazzocca et al., and sADAM9v2, a novel variant identified by the inventors of the present application. It is important to emphasize that sADAM9v2 is expressed not only by malignant prostate cancer cells but also in surrounding cells proximal to prostate cancer.

[0010] Hereinafter, the disclosure provides antibodies or antigen-binding fragments thereof that bind to sADAM9v2 protein or peptide segments thereof, characterized by a heavy chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1, a CDR2 having the amino acid sequence of SEQ ID NO: 2, and a CDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain containing, in tandem, a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0011] Preferably, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence having at least 70%, preferably at least 80%, or more preferably at least 90% identity to SEQ ID NO:7, and a light chain variable region comprising an amino acid sequence having preferably at least 70%, preferably at least 80%, or more preferably at least 90% identity to SEQ ID NO:8.

[0012] Preferably, the heavy chain comprises an amino acid sequence having at least 70%, preferably at least 80%, or more preferably at least 90% identity to SEQ ID NO:9.

[0013] Preferably, the light chain comprises an amino acid sequence having at least 70%, preferably at least 80%, or more preferably at least 90% identity to SEQ ID NO:10.

[0014] Preferably, the antibody or antigen-binding fragment is conjugated to an agent, such as a therapeutic compound, a fluorescent marker, a chemiluminescent marker, a colorimetric indicator, an enzyme label, a radioisotope, and an affinity tag.

[0015] According to a further aspect of the present disclosure, there is provided a polynucleotide encoding an antibody or antigen-binding fragment thereof of any of the above.

[0016] Another object of the present disclosure is to provide a reagent for predicting or diagnosing a sADAM9v2-associated disease, for determining the efficacy of a drug after treatment with a sADAM9v2 inhibitor, or for screening subjects for whom treatment with a sADAM9v2 inhibitor will be highly effective, said reagent comprising the above-described antibody or antigen-binding fragment thereof.

[0017] For another object of the present disclosure, there is further provided a method for predicting or diagnosing an sADAM9v2-associated disease or a predisposition to developing an sADAM9v2-associated disease in a subject, the method comprising: (a) contacting a sample isolated from the subject with any of the above-mentioned antibodies or antigen-binding fragments thereof; (b) detecting sADAM9v2 protein in the sample by detecting binding between the antibody or antigen-binding fragment thereof and the sample; and (c) comparing the level of sADAM9v2 protein in the sample with a control, wherein a higher sADAM9v2 protein level than the control indicates that the subject is suffering from or at risk of developing the disease.

[0018] Preferably, the sADAM9v2-associated disease may be a cancer that expresses sADAM9v2. Optionally, the sADAM9v2-associated disease is a cancer that overexpresses sADAM9v2.

[0019] Preferably, the cancer may be prostate cancer.

[0020] Another object of the present disclosure further provides the use of an antibody or antigen-binding fragment thereof of any of the above for the manufacture of a pharmaceutical composition for treating a sADAM9v2-associated disease.

[0021] For another object of the present disclosure, there is further provided a pharmaceutical composition comprising, as an active ingredient, an effective dose of any of the above-described antibodies or antigen-binding fragments thereof, and a pharmaceutically acceptable carrier.

[0022] As disclosed above, the present disclosure provides a monoclonal antibody developed using the specificity of soluble ADAM9 variants secreted in the vicinity of prostate cancer, which can detect the development of prostate cancer at an early stage.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0024] [Figure 1] 10 presents the results of PCR analysis revealing discrepancies between the length of ADAM9 and the previously determined length. The cell lines involved in these studies include NC (representing a negative control, no ADAM9), PC (representing a positive control for the ADAM9 gene), BPH-1 (a cell line representing benign prostatic hyperplasia-1), LNCaP (designated as a cancer cell line derived from a prostate cancer patient showing lymph node metastasis), C4-2 (a derivative of the LNCaP line characterized by androgen insensitivity), C4-2B (a subset of C4-2 cells extracted from bone metastasis in a mouse model), CWR22Rv1 (a prostate cancer cell line showing an androgen receptor mutation, an ARV7 mutation), DU145 (a prostate cancer cell line originating from a brain metastasis of a prostate cancer patient), PC3 (a prostate cancer cell line obtained from a patient with prostate cancer bone metastasis), and PC3M (a variation of PC3 cells demonstrating overt bone metastasis in an animal model). [Figure 2]

[0023] Figure 1 shows exemplary results of comprehensive sequencing of the ADAM9 gene, revealing the presence of alternative splicing variants. While ADAM9 variant 1 has been previously documented, the data also show the expression of novel ADAM9 variant 2, which has not been depicted in any previous report. [Figure 3] Figure 1 shows the exclusive identification of sADAM9 in tumor cells and adjacent peripheral cells of carcinomas. This illustration provides protein expression analysis of ADAM9 in LN (LNCaP), CWR (CWR22rv1), PC3, and DU145 cells, using both whole cell lysates and proteins isolated from culture medium (conditioned medium). Furthermore, to confirm whether sADAM9 is secreted by tumor-associated peripheral cells, cells were obtained from benign (WHN) and tumor (WHC) areas of a prostate cancer patient. [Figure 4] FIG. 1 shows that when ELISA was used to detect ADAM9 expression in the blood of benign prostatic hyperplasia (BPH) and prostate cancer patients, the expression of sADAM9 in the blood of cancer patients was significantly increased. [Figure 5] FIG. 1 shows sADAM9v2 expression in cancer and benign cells in three patients isolated by laser capture microdissection (LCM). [Figure 6] FIG. 1 shows that alternative splice sites are highly antigenic according to computational modeling. [Figure 7] FIG. 1 shows the results of hybridoma analysis for sADAM9v2. [Figure 8] FIG. 1 shows that sADAM9v2 improves the dose-dependent migration of prostate cancer cells in transwells, which can be inhibited by sADAM9v mAb. [Figure 9] FIG. 1 shows the migration and metastasis of prostate cancer cells induced by downstream signals of AKT activated by sADAM9. DETAILED DESCRIPTION OF THE INVENTION

[0025] Detailed Description of the Invention For purposes of description in this specification and the appended claims, the singular forms "a" and "an" include plural referents unless the content clearly dictates otherwise. Thus, for example, a reference to "a protein" includes a plurality of proteins, and a reference to "a compound" refers to a plurality of compounds. The use of "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and are not intended to be limiting. It should be further understood that where the description of various embodiments uses the term "comprising," those skilled in the art will understand that in some specific instances, the embodiments can instead be described using the words "consisting essentially of" or "consisting of."

[0026] Where a range of values ​​is provided, it is understood that each intervening integer, and each tenth of each intervening integer, between the upper and lower limits of that range, and any other stated or intervening value within the stated range, unless the context clearly dictates otherwise, is encompassed within the invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either (i) or (ii) both of those included limits are also encompassed within the invention. For example, "1 to 50" includes "2 to 25," "5 to 20," "25 to 50," "1 to 10," etc.

[0027] All publications, patents, patent applications, and other documents mentioned in this disclosure are incorporated by reference herein in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference herein for all purposes.

[0028] It is to be understood that both the foregoing general description, including the drawings, and the following detailed description are exemplary and explanatory only and are not restrictive of the present disclosure.

[0029] Technical and scientific terms used in the description herein have the meanings commonly understood by those skilled in the art, unless specifically defined otherwise.

[0030] I. sADAM9v2 Antibody or Antigen-Binding Fragment Thereof An antibody or an antigen-binding fragment thereof that binds to an sADAM9v2 protein or a partial peptide thereof, wherein the antibody or the antigen-binding fragment thereof is CDR1 comprising the amino acid sequence of SEQ ID NO: 1; CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 4; CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR3 comprising the amino acid sequence of SEQ ID NO: 6 and a light chain comprising

[0023] An antibody or antigen-binding fragment thereof is provided, comprising:

[0031] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 70%, preferably at least 80%, or more preferably at least 90% identity to SEQ ID NO:7, and a light chain variable region comprising an amino acid sequence having preferably at least 70%, preferably at least 80%, or more preferably at least 90% identity to SEQ ID NO:8.

[0032] In one embodiment, the heavy chain comprises an amino acid sequence having at least 70%, preferably at least 80%, or more preferably at least 90% identity to SEQ ID NO:9.

[0033] In one embodiment, the light chain comprises an amino acid sequence having at least 70%, preferably at least 80%, or more preferably at least 90% identity to SEQ ID NO:10.

[0034] Antibodies (used interchangeably in the plural) are immunoglobulin molecules capable of specifically binding to a target antigen (e.g., sADAM9v2 in the present disclosure) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses not only intact (i.e., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv), variants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of an immunoglobulin molecule that contains an antigen recognition site of a desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies include antibodies of any class, e.g., IgD, IgE, IgG, IgA, or IgM (or subclasses thereof); antibodies need not be of any particular class. Immunoglobulins can be assigned to various classes depending on the antibody amino acid sequence of the constant domain of their heavy chain. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the various classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known. As used herein, the term "isolated antibody" refers to an antibody that is substantially free of naturally associated molecules, i.e., molecules with which it is naturally associated, i.e., molecules with which it naturally comprises up to 20% of the dry mass of a preparation containing the antibody. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, and HPLC.

[0035] A typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are normally involved in antigen binding. The VH and VL regions can be further subdivided into regions of hypervariability, also known as "complementarity-determining regions" ("CDRs"), interspersed with more conserved regions known as "framework regions" ("FRs"). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino- to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The extent of framework regions and CDRs can be precisely identified using methods known in the art, for example, by the Kabat definition, the IMGT definition, the Chothia definition, the AbM definition, and / or the contact definition, all of which are well known in the art.See, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242; IMGT®, the international ImMunoGeneTics information system®, http: / / www.imgt.org; Lefranc, M.-P. et al., Nucleic Acids Res., 27:209-212 (1999); Ruiz, M. et al., Nucleic Acids Res., 28:219-221 (2000); Lefranc, M.-P., Nucleic Acids Res., 29:207-209 (2001); Lefranc, M.-P., Nucleic Acids Res., 31:307-310 (2003); Lefranc, M.-P. et al., In Silico Biol., 5, 0006 (2004) [Epub], pp. 5:45-60 (2005), Lefranc, M.-P. et al., Nucleic Acids Res., 33: D593-597 (2005), Lefranc, M.-P. et al., Nucleic Acids Res., 37: D1006-1012 (2009), Lefranc, M.-P. et al., Nucleic Acids Res., 43:D413-422 (2015), Chothia et al. (1989) Nature 342:877, Chothia. C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948, and Almagro, J. Mol. Recognit. 17:132-143 (2004). As used herein, CDRs may refer to CDRs defined by any method known in the art. Two antibodies having the same CDRs mean that the two antibodies have the same amino acid sequence of their CDRs as determined by the same method, for example, the IMGT definition.

[0036] In some embodiments, the isolated anti-sADAM9v2 antibodies described herein can bind to and inhibit sADAM9v2 activity by at least 50% (e.g., 60%, 70%, 80%, 90%, 95%, or more). The apparent inhibition constant (Kiapp or Ki,app), which provides a measure of inhibitor potency, is related to the concentration of inhibitor required to reduce enzyme activity and is independent of enzyme concentration. The inhibitory activity of the anti-sADAM9v2 antibodies described herein can be determined by routine methods known in the art.

[0037] Any of the antibodies described herein can be either monoclonal or polyclonal. A "monoclonal antibody" refers to a homogeneous antibody population, while a "polyclonal antibody" refers to a heterogeneous antibody population. These two terms do not limit the source of the antibody or the manner in which it is made.

[0038] In some embodiments, the anti-sADAM9v2 antibodies described herein bind to the same epitope on the sADAM9v2 antigen as a reference antibody disclosed herein, or compete with the reference antibody for binding to the sADAM9v2 antigen. "Epitope" refers to the site on a target compound bound by an antibody, e.g., a Fab or a full-length antibody. An epitope can be linear and typically 6-15 amino acids in length. Alternatively, an epitope can be conformational. An antibody that binds to the same epitope as a reference antibody described herein can bind the exact same epitope as the reference antibody or a substantially overlapping epitope (e.g., containing fewer than three non-overlapping amino acid residues, fewer than two non-overlapping amino acid residues, or only one non-overlapping amino acid residue). Whether two antibodies compete with each other for binding to a cognate antigen can be determined by competition assays well known in the art. Such antibodies can be identified as known to those of skill in the art, e.g., by having substantially similar structural characteristics (e.g., complementarity determining regions) and / or by assays known in the art. For example, a competition assay can be performed using one of the reference antibodies to determine whether a candidate antibody binds to the same epitope as the reference antibody or competes for its binding to the sADAM9v2 antigen.

[0039] In one example, the antibody used in the methods described herein can be a humanized antibody. Humanized antibodies refer to forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody), e.g., mouse, rat, or rabbit, having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine or optimize antibody performance. Generally, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions being human immunoglobulin consensus sequences. A humanized antibody optimally also comprises at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. The antibody may have an Fc region modified as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) that are altered relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody. Humanized antibodies may also undergo affinity maturation.

[0040] In some embodiments, the anti-sADAM9v2 antibodies described herein specifically bind to their corresponding target antigen or epitope thereof. An antibody that "specifically binds" to an antigen or epitope is a term well understood in the art. A molecule is said to exhibit "specific binding" when it reacts with a particular target antigen more frequently, rapidly, for a longer duration, and / or with higher affinity than to alternative targets. An antibody "specifically binds" to a target antigen or epitope when it binds with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to an antigen (e.g., human sADAM9v2) or an antigenic epitope therein is an antibody that binds to this target antigen with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other antigens or other epitopes within the same antigen. It should also be understood that in this definition, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. Thus, "specific binding" or "preferential binding" does not necessarily require (but can include) exclusive binding. In some instances, an antibody that "specifically binds" to a target antigen or epitope thereof may not bind to other antigens or other epitopes within the same antigen (e.g., binding is not detectable in conventional assays).

[0041] In some embodiments, the antibodies described herein specifically bind to sADAM9v2 of a particular species (e.g., human sADAM9v2) as a relative of sADAM9v2 from other species. For example, the antibodies described herein may specifically bind to human sADAM9v2 as a relative of mouse sADAM9v2. In other embodiments, the antibodies described herein may cross-react with human sADAM9v2 and one or more sADAM9v2 from a non-human species (e.g., a non-human primate, e.g., macaque). In some embodiments, the antibodies cross-react with human and rhesus monkeys with similar binding affinity, but have significantly lower binding affinity for mouse sADAM9v2. In some embodiments, the anti-sADAM9v2 antibodies described herein have suitable binding affinity for a target antigen (e.g., human sADAM9v2) or an antigenic epitope thereof.

[0042] As used herein, "binding affinity" refers to the apparent association constant or K, which is the ratio of the association and dissociation constants, K and K, respectively. A is the dissociation constant (K D The anti-sADAM9v2 antibodies described herein have a target antigen or antigen epitope affinity of at least 10 -8 , 10 -9 , 10 -10 M, 10 -11 M or lower binding affinity (K D For example, the anti-sADAM9v2 antibody may have a 10 -9 M, 10 -10 M, or lower. An increased binding affinity may have a decreased K D A higher affinity binding of an antibody to a first antigen compared to a second antigen corresponds to a higher K (or K) value for binding to the second antigen. D a K (or a smaller K value) for binding to the first antigen DIn such cases, the antibody has specificity for a first antigen (e.g., a first protein or mimetic thereof in a first conformation) compared to a second antigen (e.g., the same first protein or mimetic thereof in a second conformation, or a second protein). In some embodiments, the anti-sADAM9v2 antibodies described herein have a higher binding affinity (higher K A or smaller K D In some embodiments, an anti-sADAM9v2 antibody may have a higher binding affinity for sADAM9v2 of a particular species (e.g., human sADAM9v2) than for sADAM9v2 from a different species (e.g., mouse). The difference in binding affinity (e.g., for specificity or other comparison) may be at least 1.5, 2, 2.5, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1,000, 5,000, 10,000, or 10 5 In some embodiments, any of the anti-sADAM9v2 antibodies may be further affinity matured to increase the binding affinity of the antibody for the target antigen or antigenic epitope thereof.

[0043] Binding affinity (or binding specificity) can be determined by a variety of methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance (SPR), fluorescence-activated cell sorting (FACS), or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for assessing binding affinity include HBS-P buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% (v / v) surfactant P20) as well as PBS buffer (10 mM PO4 -3 , 137 mM NaCl, and 2.7 mM KCl). These techniques can be used to measure the concentration of bound protein as a function of target protein concentration. The concentration of bound protein ([bound]) is generally related to the concentration of free target protein ([free]) by the following equation: [Bound]=[Free] / (Kd+[Free])

[0044] While it is not always necessary to make an exact determination of the K, it may be sufficient to obtain, for example, a quantitative measure of affinity determined using methods such as ELISA or FACS analysis that is proportional to the K and can therefore be used to compare, e.g., determine whether a higher affinity is, e.g., two-fold higher, to obtain a qualitative measure of affinity, or to obtain an indication of affinity, for example, by activity in a functional assay, e.g., an in vitro or in vivo assay.

[0045] In some cases, the variation of the amino acid residue can be a conservative amino acid residue substitution. As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to references that compile such methods, for example, methods for modifying polypeptide sequences known to those skilled in the art, such as those found in Molecular Cloning: A Laboratory Manual, edited by J. Sambrook et al., 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, or Current Protocols in Molecular Biology, edited by E.M. Ausubel et al., John Wiley & Sons, Inc., New York.

[0046] In some embodiments, the heavy chain of any of the anti-sADAM9v2 antibodies described herein may further comprise a heavy chain constant region (5CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region can be of any suitable origin, for example, human, mouse, rat, or rabbit. In one specific example, the heavy chain constant region is from human IgG (gamma heavy chain), for example, IgG1, IgG2, or IgG4. In one example, the heavy chain constant region is of the subclass IgG1.

[0047] The light chain of any of the anti-sADAM9v2 antibodies described herein can further comprise a light chain constant region (CL), which can be any CL known in the art. In some examples, the CL is a kappa light chain. In other examples, the CL is a lambda light chain. Antibody heavy and light chain constant regions are well known in the art, such as those provided in the IMGT database (www.imgt.org) or www.vbase2.org / vbstat.php, both of which are incorporated herein by reference.

[0048] As described herein, anti-sADAM9v2 antibodies can be any antibody form, including, but not limited to, an intact (i.e., full-length) antibody, an antigen-binding fragment thereof (Fab, Fab', F(ab')2, Fv, etc.), a single-chain antibody, a bispecific antibody, or a nanobody.

[0049] II. Preparation of anti-sADAM9v2 antibody Antibodies capable of binding to sADAM9v2 described herein can be produced by any method known in the art, see, e.g., Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

[0050] In some embodiments, antibodies specific to a target antigen (e.g., sADAM9v2) can be produced by conventional hybridoma technology. A full-length target antigen or a fragment thereof, optionally coupled to a carrier protein such as KLH, can be used to immunize a host animal to produce antibodies that bind the antigen. As further described herein, the route and schedule of immunization of a host animal generally conforms to established, conventional techniques for stimulating and producing antibodies. General techniques for producing murine, humanized, and human antibodies are known in the art and are described herein. It is contemplated that any mammalian subject, including humans, or antibody-producing cells therefrom, can be engineered to serve as the basis for the generation of mammalian, including human hybridoma cell lines. Typically, a host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantarly, and / or intradermally with an amount of an immunogen, including those described herein.

[0051] If desired, the antibody of interest (monoclonal or polyclonal) (e.g., produced by a hybridoma) may be sequenced, and the polynucleotide sequence may then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest may be maintained in a vector in a host cell, which may then be expanded and frozen for future use. Alternatively, the polynucleotide sequence may be used for genetic engineering to "humanize" the antibody or to improve affinity (affinity maturation) or other characteristics of the antibody. For example, if the antibody is to be used in human clinical trials and treatments, the constant region may be engineered to more resemble human constant regions to avoid an immune response. It may be desirable to genetically engineer the antibody sequence to obtain higher affinity for the target antigen and greater efficacy in inhibiting the activity of sADAM9v2. It will be apparent to those skilled in the art that one or more polynucleotide changes can be made to an antibody and still maintain its binding specificity for the target antigen.

[0052] In other embodiments, fully human antibodies can be obtained by using commercially available mice engineered to express specific human immunoglobulin proteins. Transgenic animals designed to generate a more desirable (e.g., fully human antibodies) or more robust immune response can also be used to generate humanized or human antibodies. Examples of such technologies are the Xenomouse® from Amgen, Inc. (Fremont, CA) and the HuMAb-Mouse® and TC Mouse® from Medarex, Inc. (Princeton, NJ), or the H2L2 mouse from Habour Antibodies BV (The Netherlands). In another alternative, antibodies can be produced recombinantly by phage display or yeast technology. See, e.g., U.S. Patent Nos. 5,565,332, 5,580,717, 5,733,743, and 6,265,150, and Winters et al. (1994) Anno. Rev. Immunol. 12:433-455. Alternatively, using phage display technology (McCafferty et al. (1990) Nature 348:552-553), human antibodies and antibody fragments can be produced in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.

[0053] Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared by routine methods. For example, F(ab')2 fragments can be generated by pepsin digestion of antibody molecules and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragment. Genetically engineered antibodies, such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bispecific antibodies, can be produced, for example, through conventional recombinant techniques. In one example, DNA encoding monoclonal antibodies specific for a target antigen can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into one or more expression vectors, which can then be transfected into host cells that do not otherwise produce immunoglobulin proteins, such as Escherichia coli (E. coli) cells, simian COS cells, Chinese hamster ovary (CHO) cells, human HEK293 cells, or myeloma cells, resulting in the synthesis of monoclonal antibodies in the recombinant host cells. See, e.g., PCT Publication WO 87 / 04462. The DNA can then be modified, e.g., by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences, as described by Morrison et al. (1984) Proc. Nat. Acad. Sci. 81:6851, or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this manner, engineered antibodies, e.g., "chimeric" or "hybrid" antibodies, with the binding specificity of a target antigen can be prepared.

[0054] Single-chain antibodies can be prepared by recombinantly linking a nucleotide sequence encoding a heavy chain variable region with a nucleotide sequence encoding a light chain variable region, preferably incorporating a flexible linker between the two variable regions.

[0055] Antibodies obtained according to the methods known in the art and described herein can be characterized using methods well known in the art. For example, one method is to identify the epitope to which an antigen binds, or to perform "epitope mapping." Numerous methods are known in the art for mapping and characterizing the location of epitopes in proteins, including analysis of the crystal structure of antibody-antigen complexes, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, "Using Antibodies, a Laboratory Manual," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. In one example, epitope mapping can be achieved using H / D-Ex (hydrogen-deuterium exchange) coupled with proteolysis and mass spectrometry. In a further example, epitope mapping can be used to determine the sequence to which an antibody binds. Epitopes can be linear epitopes, i.e., contained in a single stretch of amino acids, or conformational epitopes formed by tertiary interactions of amino acids that are not necessarily contained in a single stretch (linear sequence of primary structure). Peptides of various lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinantly) and used in antibody-based binding assays. In another example, the epitope to which an antibody binds can be determined in a systematic screen by using overlapping peptides derived from the target antigen sequence and determining binding by the antibody. In a gene fragment expression assay, the open reading frame encoding the target antigen is fragmented either randomly or by a specific gene construct, and the reactivity of the expressed antigen fragment with the antibody being tested is determined. Gene fragments can be generated, for example, by PCR, and then transcribed and translated into protein in vitro in the presence of radioactive amino acids. Binding of the antibody to the radiolabeled antigen fragment is then determined by immunoprecipitation and gel electrophoresis.Specific epitopes can also be identified by using large libraries of random peptide sequences (phage libraries) displayed on the surface of phage particles. Alternatively, a defined library of overlapping peptide fragments can be tested for binding with a test antibody in a simple binding assay. In a further example, mutagenesis of the antigen-binding domain, domain swapping experiments, and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and / or necessary for epitope binding. For example, domain swapping experiments can be performed using mutants of the target antigen in which various fragments of the sADAM9v2 polypeptide are replaced (swapped) with sequences from a closely related but antigenically distinct protein (e.g., CD-28 protein). The importance of specific antigen fragments in antibody binding can be assessed by assessing the binding of the antibody to mutant sADAM9v2. Alternatively, a competition assay can be performed using other antibodies known to bind the same antigen to determine whether the antibody binds to the same epitope as other antibodies. Competition assays are well known to those skilled in the art.

[0056] In some examples, anti-sADAM9v2 antibodies are prepared by recombinant techniques, as exemplified below. Nucleic acids encoding the heavy and light chains of the anti-sADAM9v2 antibodies described herein can be cloned into a single expression vector, with each nucleotide sequence operably linked to an appropriate promoter. In one example, the nucleotide sequences encoding the heavy and light chains are operably linked to distinct promoters. Alternatively, the nucleotide sequences encoding the heavy and light chains can be operably linked to a single promoter so that both the heavy and light chains are expressed from the same promoter. If necessary, an internal ribosome entry site (IRES) can be inserted between the coding sequences for the heavy and light chains.

[0057] In some instances, the nucleotide sequences encoding the two chains of an antibody can be cloned into two vectors and introduced into the same or different cells. If the two chains are expressed in different cells, each can be isolated from the host cell in which it is expressed, and the isolated heavy and light chains can be mixed and incubated under appropriate conditions to allow antibody formation.

[0058] Generally, using methods known in the art, a nucleic acid sequence encoding one or all chains of an antibody can be operably linked to a suitable promoter and cloned into a suitable expression vector. For example, the nucleotide sequence and the vector can be contacted with a restriction enzyme under appropriate conditions to create complementary ends on each molecule that can pair with each other and be joined together via a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the ends of the gene. These synthetic linkers contain nucleic acid sequences that correspond to specific restriction sites in the vector. The choice of expression vector / promoter will depend on the type of host cell used to produce the antibody.

[0059] Regulatable promoters can be used, including those that use the lac repressor from Escherichia coli as a transcriptional modulator to control transcription from mammalian cell promoters containing the lac operator [Brown, M. et al., Cell, 49:603-612 (1987)] or the tetracycline repressor (tetR) [Gossen, M. and Bujard, H., Proc. Natl. Acad. Sci. USA, 89:5547-555115 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P. et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimers, VP16, or p65 using astradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech, and Ariad, among others.

[0060] Regulatable promoters containing a repressor in conjunction with an operon can be used. In one embodiment, the lac repressor from Escherichia coli can function as a transcriptional modulator to regulate transcription from mammalian cell promoters carrying the lac operator [M. Brown et al., Cell, 49:603-612 (1987)]. Gossen and Bujard (1992) [M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)] combined the tetracycline repressor (tetR) with a transcriptional activator (VP 16) to create a tetR-mammalian cell transcriptional activator fusion protein, tTa (tetR-VP 16), which was combined with tetO, carrying a minimal promoter derived from the human cytomegalovirus (hCMV) promoter, to create a tetR-tet operator system for controlling gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. The tetracycline repressor (tetR) alone, rather than a tetR-mammalian cell transcription factor fusion derivative, can function as a potent transmodulator for regulating gene expression in mammalian cells when the tetracycline operator is appropriately positioned downstream of the TATA element of the CMV1E promoter (Yao et al., Human Gene Therapy). One particular advantage of this tetracycline-inducible switch is that it does not require the use of a tetracycline repressor mammalian cell transactivator or repressor fusion protein, which in some cases may be toxic to cells, to achieve its regulatable effect (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)).

[0061] Additionally, vectors can contain, for example, some or all of the following: a selectable marker gene, e.g., a neomycin gene for selection of stable or transient transfectants in mammalian cells; an enhancer / promoter sequence from the immediate-early gene of human CMV for high-level transcription; a transcription termination and RNA processing signal from SV40 for mRNA stability; an SV40 polyoma origin of replication and ColE1 for proper episomal replication; an internal ribosome binding site (IRES); a versatile multiple cloning site; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for generating vectors containing transgenes are known and available in the art. Examples of polyadenylation signals useful for carrying out the methods described herein include, but are not limited to, the human collagen I polyadenylation signal, the human collagen II polyadenylation signal, and the SV40 polyadenylation signal.

[0062] One or more vectors (e.g., expression vectors) containing nucleic acids encoding any of the antibodies can be introduced into suitable host cells for antibody production. The host cells can be cultured under appropriate conditions for expression of the antibody or any polypeptide chain thereof. Such antibodies or polypeptide chains thereof can be recovered from the cultured cells (e.g., from the cells or culture supernatant) by conventional methods, such as affinity purification. If desired, the antibody polypeptide chains can be incubated under appropriate conditions for a suitable period of time to allow antibody production.

[0063] In some embodiments, the methods for preparing the antibodies described herein include a recombinant expression vector encoding both the heavy and light chains of an anti-sADAM9v2 antibody, also as described herein. The recombinant expression vector can be introduced into suitable host cells (e.g., dhfr-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection. Positively transformed host cells can be selected and cultured under appropriate conditions to allow expression of the two polypeptide chains that form the antibody, which can be recovered from the cells or culture medium. If necessary, the two chains recovered from the host cells can be incubated under appropriate conditions to allow antibody formation.

[0064] In one example, two recombinant expression vectors are provided, one encoding the heavy chain of an anti-sADAM9v2 antibody and the other encoding the light chain of an anti-sADAM9v2 antibody, and both recombinant expression vectors can be introduced into suitable host cells (e.g., dhfr-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection.

[0065] Alternatively, each of the expression vectors can be introduced into a suitable host cell. Positive transformants can be selected and cultured under appropriate conditions to allow expression of the antibody polypeptide chains. If two expression vectors are introduced into the same host cell, the antibody produced therein can be recovered from the host cell or culture medium. If necessary, the polypeptide chains can be recovered from the host cell or culture medium and then incubated under appropriate conditions to allow antibody formation. If two expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cell or corresponding culture medium. The two polypeptide chains can then be incubated under appropriate conditions for antibody formation.

[0066] Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography using a Protein A, Protein G, or Protein L coupled matrix.

[0067] Any of the nucleic acids encoding the heavy chain, light chain, or both of the anti-sADAM9v2 antibodies described herein, vectors (e.g., expression vectors) containing each, and host cells containing the vectors are within the scope of the present disclosure.

[0068] III. Pharmaceutical Compositions The antibodies described herein, and nucleic acids or nucleic acid sets encoding them, vectors containing such, or host cells containing the vectors, can be mixed with a pharmaceutically acceptable carrier (excipient) to form a pharmaceutical composition for use in treating the target disease. By "acceptable," it is meant that the carrier must be compatible with (and preferably be able to stabilize) the active ingredients of the composition and not deleterious to the subject being treated. Pharmaceutically acceptable excipients (carriers) include buffers, which are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy, 20th ed. (2000) Lippincott Williams and Wilkins, ed. K. E. Hoover.

[0069] Pharmaceutical compositions containing the anti-sADAM9v2 antibodies disclosed herein may further comprise a suitable buffer. A buffer is a weak acid or salt used to maintain the pH of a solution near a selected value after the addition of another acid or base. In some examples, the buffers disclosed herein can be buffers that can maintain physiological pH despite changes in carbon dioxide concentration (produced by cellular respiration). Exemplary buffers include, but are not limited to, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, Dulbecco's phosphate-buffered saline (DPBS) buffer, or phosphate-buffered saline (PBS) buffer. Such buffers can include disodium hydrogen phosphate and sodium chloride, or potassium dihydrogen phosphate and potassium chloride.

[0070] In some embodiments, the buffering agent in the pharmaceutical compositions described herein may maintain a pH value of about 5 to 8. For example, the pH of the pharmaceutical composition may be about 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In other examples, the pharmaceutical composition may have a pH value below 7, e.g., about 7, 6.8, 6.5, 6.3, 6, 5.8, 5.5, 5.3, or 5.

[0071] The pharmaceutical compositions described herein contain one or more suitable salts. Salts are ionic compounds that can be formed by the neutralization reaction of an acid and a base. (Skoog, DA; West, D M.; Holler, JF; Crouch, SR (2004). Chapters 14-16, Fundamentals of Analytical Chemistry (8th Edition)). Salts are composed of a related number of cations (positively charged ions) and anions (negative ions) such that the product is electrically neutral (no net charge). As described herein, ions are atoms or molecules that have gained or lost monovalent or multivalent electrons, giving the ion a net positive or negative charge. If a chemical species has more protons than electrons, it possesses a net positive charge. If there are more electrons than protons, the species has a negative charge.

[0072] As used herein, a cation (+) is an ion that has fewer electrons than protons, giving it a positive charge. (Douglas W. Haywick, (2007-2008). "Elemental Chemistry"). A cation with one positive charge can be called a monovalent cation, and a cation with multiple positive charges can be called a polyvalent or multivalent cation. A non-limiting example of a monovalent cation is hydrogen (H + ), sodium (Na + ), potassium (K + ), ammonium (NH 4+ ), lithium (Li + ), Copper monovalent (Cu + ), silver (Ag + ), etc. Non-limiting examples of polyvalent cations include magnesium (Mg 2+ ), calcium (Ca 2+ ), barium (Ba 2+ ), Beryllium (Be 2+ ), copper divalent (Cu 2+ ), iron (Fe 2+ ), iron(III) 3+ ), lead(II)(Pb 2+ ), lead(IV)(Pb 4+ ), manganese(II)(Mn 2+ ), strontium (Sr 2+ ), tin(IV) (Sn 4+ ), zinc (Zn 2+ ) etc.

[0073] As used herein, an anion is an ion that has more electrons than protons, giving it a net negative charge. A non-limiting example of an anion is azide (N 3- ), bromide ion (Br - ), chloride ions (Cl - l), fluoride ion (F - ), hydride (H - ), iodide ion (I - ), Nitride (N - ), oxide (O 2- ), sulfide (S2- ), carbonate ions (CO3 2- ), bicarbonate ion (HCO3 - ), hydrogen sulfate ion (HSO4 - ), hydroxide ion (OH - ), dihydrogen phosphate ion (H2PO4 - ), sulfate ions (SO4 2- ), sulfite ions (SO3 2- ), silicate ions (SiO3 2- ) etc.

[0074] Suitable salts for use in the pharmaceutical compositions described herein may contain a monovalent cation and a monovalent or polyvalent anion. Alternatively, salts for use in the pharmaceutical compositions described herein may contain a monovalent or polyvalent cation and a monovalent anion. Exemplary salts include, but are not limited to, potassium chloride (KCl), sodium chloride (NaCl), calcium chloride (CaCl), magnesium chloride (MgCl), magnesium sulfate (MgSO), sodium bicarbonate (NaHCO), ammonium sulfate ((NH)SO), calcium carbonate (CaCO), or a combination thereof.

[0075] The pharmaceutical compositions described herein contain one or more suitable surface-active agents, such as surfactants. A surfactant is a compound that reduces the surface tension (or interfacial tension) between two liquids, between a gas and a liquid, or between a liquid and a solid. Surfactants can act as detergents, wetting agents, emulsifiers, foaming agents, and dispersing agents. Suitable surfactants include, among others, nonionic agents such as polyoxyethylene sorbitan (e.g., Tween™ 20, 40, 60, 80, or 85) and other sorbitans (e.g., Span™ 20, 40, 60, 80, or 85). Compositions with surfactants conveniently contain 0.05-5% surfactant, and can be 0.1-2.5%. It will be understood that other ingredients, such as mannitol or other pharmaceutically acceptable vehicles, can be added if desired.

[0076] Pharmaceutical compositions containing anti-sADAM9v2 described herein can include one or more amino acids. Exemplary amino acids include, but are not limited to, glycine, histidine, or arginine.

[0077] The pharmaceutical composition may also contain one or more antioxidants. As used herein, an antioxidant is an agent that prevents or delays the oxidative degradation of an active ingredient contained in the composition. As used herein, an antioxidant may be a phenolic antioxidant (sometimes referred to as a true antioxidant), a reducing agent, or a chelating agent. Phenolic antioxidants are sterically hindered phenols that react with free radicals and block chain reactions. Reducing agents are compounds that have a lower redox potential and are therefore more easily oxidized than the drugs they are intended to protect. Reducing agents scavenge oxygen from the medium, thus delaying or preventing the oxidation of the drug. Chelating agents are sometimes referred to as antioxidant synergists. Metal ions, such as Co, 2+ , Cu 2+ , Fe 3+ , Fe 2+ , and Mn 2+ Chelating agents shorten the induction period and increase the rate of oxidation. Trace amounts of these metal ions are frequently introduced into drug products during manufacturing. Chelating agents do not possess antioxidant activity themselves, but they enhance the action of phenolic antioxidants by reacting with catalytic metal ions, deactivating them.

[0078] The pharmaceutical compositions described herein may also include sugar derivatives. As used herein, sugar derivatives include sugars and organic compounds derived from sugars. In some cases, the sugar derivative may be a non-reducing sugar, sugar alcohol, polyol, disaccharide, or polysaccharide.

[0079] The pharmaceutical composition used in the present method can contain a pharmaceutically acceptable carrier, excipient, or stabilizer in the form of a lyophilized formulation or aqueous solution. (Remington: The Science and Practice of Pharmacy, 20th ed. (2000) Lippincott Williams and Wilkins, ed. K. E. Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations used, and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); polypeptides of low molecular weight (less than about 10 residues); proteins, such as serum alcohols. The composition may include immunoglobulin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0080] In some instances, the pharmaceutical compositions described herein include liposomes containing the antibody (or a nucleic acid encoding it), which can be prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985), Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980), and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse-phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.

[0081] The antibody or nucleic acid encoding it may also be entrapped in microcapsules, prepared, for example, by coacervation techniques, or by interfacial polymerization, e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacylated) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions. Such techniques are known in the art; see, e.g., Remington, The Science and Practice of Pharmacy, 20th ed., Mack Publishing (2000).

[0082] In other examples, the pharmaceutical compositions described herein can be formulated in a sustained-release form. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vnlalcohol)), polylactide (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L20 glutamic acid, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0083] In other examples, the pharmaceutical compositions described herein can be formulated in sustained release formats that affect selective binding to tissues or tumors by implementing specific protease biology technologies, such as peptide masking of the antigen binding site of an antibody to allow selective protease cleavability by one or more proteases in the tumor microenvironment, e.g., Probody™ or Conditionally Active Biologics™. Activation can be formulated to be reversible in the normal microenvironment.

[0084] Pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes. Therapeutic antibody compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0085] The pharmaceutical compositions described herein may be in unit dosage form, such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral, or rectal administration, or administration by inhalation or insufflation.

[0086] To prepare solid compositions, such as tablets, the active ingredient can be mixed with a pharmaceutical carrier, such as conventional tableting ingredients, such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gums, and other pharmaceutical diluents, such as water, to form a solid preformulation composition containing a homogeneous mixture of the compound of the present invention or a non-toxic, pharmaceutically acceptable salt thereof. When these preformulation compositions are referred to as homogeneous, this means that the active ingredient is evenly dispersed throughout the composition so that the composition can be easily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. Tablets or pills of the novel compositions can be coated or otherwise compounded to provide a dosage form offering the advantage of prolonged action. For example, a tablet or pill can include an inner dosage and an outer dosage component, the latter in the form of an envelope over the former. The two components can be separated by an enteric layer, which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, including a number of polymeric acids and mixtures of polymeric acids with such materials, for example, shellac, cetyl alcohol, and cellulose acetate.

[0087] Suitable emulsions can be prepared using commercially available fat emulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™, and Lipiphysan™. The active ingredient can be dissolved in a premixed emulsion composition, or in an emulsion formed when mixed with an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and a phospholipid (e.g., egg phospholipid, soybean phospholipid, or soybean lecithin) and water. It will be appreciated that other ingredients, such as glycerol or glucose, can be added to adjust the isotonicity of the emulsion. Suitable emulsions typically contain up to 20% oil, e.g., 5-20%. Fat emulsions contain 0.1-1.0 μm, particularly 0.1-0.5 μm, fat droplets and can have a pH in the range of 5.5-8.0. Emulsion compositions can be prepared by mixing antibodies with Intralipid™ or its components (soybean oil, egg phospholipids, glycerol, and water).

[0088] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof, as well as powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions, preferably in sterile pharmaceutically acceptable solvents, may be nebulized by the use of gases. Nebulized solutions may be breathed directly from the nebulizing device, or the nebulizing device may be attached to a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from a device that delivers the formulation in an appropriate manner.

[0089] IV. Therapeutic Applications Any of the antibodies described herein, as well as nucleic acids or nucleic acid sets encoding them, vectors containing such, or host cells containing the vectors, are useful for treating sADAM9v2-mediated disorders. As used herein, an sADAM9v2-mediated disease refers to any condition associated with increased levels of sADAM9v2 or increased sensitivity to sADAM9v2. Non-limiting examples of sADAM9v2-mediated diseases include prostate cancer.

[0090] To practice the methods disclosed herein, an effective amount of the pharmaceutical compositions described herein can be administered to a subject (e.g., a human) in need of treatment by an appropriate route, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, intramuscularly, intraperitoneally, intracerebrospinal, subcutaneously, intra-articularly, intrasynovially, intrathecally, orally, by inhalation, or topically. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations can be nebulized directly, and lyophilized powders can be nebulized after reconstitution. Alternatively, the antibodies described herein can be aerosolized using fluorocarbon formulations and metered-dose inhalers, or inhaled as a lyophilized and milled powder.

[0091] The subject treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice, and rats. A human subject in need of treatment can be a human patient with, at risk for, or suspected of having an inflammatory disease, autoimmune disease, cancer, infectious disease, or other disorder requiring modulation of the immune response. A subject with a target disease or disorder can be identified by routine medical tests, such as laboratory tests, organ function tests, CT scans, or ultrasound. A subject suspected of having any of such target diseases / disorders can exhibit one or more symptoms of the disease / disorder. A subject at risk for a disease / disorder can be a subject with one or more risk factors for the disease / disorder.

[0092] As used herein, "effective amount" refers to the amount of each active agent, either alone or in combination with one or more other active agents, required to confer a therapeutic effect on a subject. In some embodiments, the therapeutic effect is a reduction in sADAM9v2 activity.

[0093] As used herein, "overexpress" refers to cancer cells having significantly higher surface expression of sADAM9v2 than normal cells.

[0094] Determining whether a certain amount of antibody has achieved a therapeutic effect will be apparent to one skilled in the art. As will be understood by those skilled in the art, an effective amount will vary depending on the particular condition being treated, the severity of the condition, individual patient parameters including age, physical condition, height, sex, and weight, the duration of treatment, the nature of concurrent treatment (if any), the specific route of administration, and similar factors within the knowledge and professional opinion of a health professional. These factors are well known to those skilled in the art and can be addressed with no more than routine experimentation.

[0095] In general, it is preferred to use the maximum dose of any individual component or combination thereof, that is, the highest safe dose according to sound medical judgment.

[0096] Empirical considerations, such as half-life, generally contribute to determining the dosage. For example, antibodies compatible with the human immune system, such as humanized or fully human antibodies, can be used to extend the half-life of the antibody and prevent it from being attacked by the host's immune system. The frequency of administration can be determined and adjusted during treatment and is generally, but not necessarily, based on the treatment and / or suppression and / or remission and / or delay of the target disease / disorder. Alternatively, a sustained continuous release formulation of the antibody may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0097] In one example, the dosage of the antibodies described herein can be empirically determined in an individual given one or more doses of the antibody. The individual is given increasing dosages of the antagonist. Disease / disorder indicators can be followed to assess the effectiveness of the antagonist.

[0098] Generally, for administration of any of the antibodies described herein, an initial candidate dosage can be about 2 mg / kg. For purposes of the present disclosure, a typical once-daily, once-weekly, every two weeks, or every three weeks dosage can range from approximately any of 0.1 μg / kg to 3 μg / kg to 30 μg / kg to 100 μg / kg to 300 μg / kg to 0.6 mg / kg, 1 mg / kg, 3 mg / kg, 10 mg / kg, 30 mg / kg, 100 mg / kg, or more, depending on the factors mentioned above. For repeated administrations over several days, weeks, months, or longer, depending on the condition, treatment is sustained until a desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved to alleviate the target disease or disorder or its symptoms. An exemplary dosing regimen involves administering an initial dose of about 3 mg / kg every three weeks, followed by a maintenance dose of about 1 mg / kg of antibody every six weeks, or a maintenance dose of about 1 mg / kg every three weeks. However, other dosing regimens may be useful depending on the pattern of pharmacokinetic decay the practitioner desires to achieve. For example, dosing at 1 mg / kg every three weeks in combination with at least one additional immunotherapeutic agent is contemplated. In some embodiments, dosages ranging from about 3 μg / mg to about 3 mg / kg (such as about 3 μg / mg, about 10 μg / mg, about 30 μg / mg, about 100 μg / mg, about 300 μg / mg, about 1 mg / kg, and about 3 mg / kg) may be used. In some embodiments, the frequency of administration is once a week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks, or once a month, every 2 months, or every 3 months, or longer. The progress of this treatment is easily monitored by conventional techniques and assays. The dosing regimen (including the antibody used) can be varied over time.

[0099] In some embodiments, a normal weight adult patient may receive a dose ranging from about 0.1 to 5.0 mg / kg. In some instances, the dosage of the anti-sADAM9v2 antibodies described herein may be 10 mg / kg. The particular dosing regimen, i.e., dose, timing, and repetition, will depend on the particular individual and their medical history, as well as the characteristics of the individual drug (such as the drug's half-life and other considerations known in the art).

[0100] For purposes of this disclosure, appropriate dosages of the antibodies described herein will depend on the specific antibody, antibodies, and / or non-antibody peptide (or composition thereof) used, the type and severity of the disease / disorder, whether the antibody is being administered for preventative or therapeutic purposes, previous treatments, the patient's medical history and response to antagonists, and the discretion of the attending physician. Typically, a clinician will administer the antibody until a dosage is reached that achieves the desired result. In some embodiments, the desired result is a reduction in tumor size, an increase in progression-free survival, and / or overall survival. Methods for determining whether a dosage has produced a desired result will be apparent to those of skill in the art. Administration of one or more antibodies can be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those of skill in the art. Administration of the antibody can be essentially continuous over a preselected period of time, for example, either before, during, or after the onset of the target disease or disorder, or can be in a series of spaced doses.

[0101] As used herein, the term "treating" refers to the application or administration of a composition containing one or more active agents to a subject with a target disease or disorder, a symptom of a disease / disorder, or a predisposition to a disease / disorder, with the intent to cure, heal, alleviate, relieve, alter, treat, ameliorate, improve, or affect the disorder, symptom of the disease, or predisposition to the disease or disorder. Alleviating a target disease / disorder includes delaying the onset or progression of the disease or reducing the severity of the disease.

[0102] Alleviating a disease does not necessarily require a curative result. As used herein, "delaying" the onset of a target disease or disorder means postponing, hindering, slowing, retarding, stabilizing, and / or postponing the progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or the individual being treated. A method of "delaying" or alleviating the onset of a disease, or delaying the onset of a disease, is a method that reduces the probability of developing one or more symptoms of the disease within a given time frame and / or reduces the severity of the symptoms within a given time frame, compared to not using the method. Such comparisons are typically based on clinical studies using a sufficient number of subjects to provide statistically significant results.

[0103] In some embodiments, the antibodies described herein are administered to a subject in need of treatment in an amount sufficient to inhibit the activity of the target antigen in vivo by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more). In other embodiments, the antibodies are administered in an amount effective to reduce the activity level of the target antigen by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more).

[0104] The pharmaceutical composition can be administered to a subject using conventional methods known to those skilled in the medical field, depending on the type of disease or the site of the disease to be treated. The composition can also be administered via other conventional routes, for example, parenterally, topically, orally, by inhalation spray, rectally, nasally, bucally, vaginally, or via an implanted reservoir. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intraperitoneal, intratumoral, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. Furthermore, it can be administered to a subject via an injectable depot administration route, for example, a 1-, 3-, or 6-month depot injection, or using biodegradable materials and methods.

[0105] Injectable compositions may contain a variety of carriers, such as vegetable oils, dimethyl lactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injections, water-soluble antibodies can be administered by infusion, in which a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. For intramuscular preparations, for example, a sterile formulation of a suitable soluble salt form of the antibody can be administered in a pharmaceutical excipient, such as water for injection, 0.9% saline, or 5% glucose solution.

[0106] In one embodiment, the antibody is administered via a site-specific or targeted local delivery technique. Examples of site-specific or targeted local delivery techniques include various implantable antibody depot sources or local delivery catheters, such as infusion catheters, indwelling catheters, or needle catheters, artificial vascular grafts, adventitial wraps, shunts, and stents, or other implantable devices, site-specific carriers, direct injection, or direct application. See, e.g., PCT Publication WO 00 / 53211 and U.S. Patent No. 5,981,568.

[0107] Targeted delivery of therapeutic compositions containing antisense polynucleotides, expression vectors, or subgenomic polynucleotides can also be used.Receptor-mediated DNA delivery techniques are described, for example, in Findeis et al., Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods and Applications of Direct Gene Transfer (ed. J. A. Wolff) (1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA (1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338.

[0108] Therapeutic compositions containing polynucleotides (e.g., encoding the antibodies described herein) are administered in the range of about 100 ng to about 200 mg of DNA for local administration in gene therapy protocols. In some embodiments, concentrations of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA, or higher, can also be used during gene therapy protocols.

[0109] The therapeutic polynucleotides and polypeptides described herein can be delivered using gene delivery vehicles. Gene delivery vehicles can be of viral or non-viral origin (see generally Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters and / or enhancers. Expression of the coding sequences can be either constitutive or regulated.

[0110] Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90 / 07936, PCT Publication No. WO 94 / 03622, PCT Publication No. WO 93 / 25698, PCT Publication No. WO 93 / 25234, PCT Publication No. WO 93 / 11230, PCT Publication No. WO 93 / 10218, PCT Publication No. WO 91 / 02805, U.S. Pat. Nos. 5,219,740 and 4,777,127, British Patent No. 2,200,651, and European Patent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki Forest virus (ATCC VR-67, ATCC VR-1247), Ross River virus (ATCC VR-373, ATCC VR-1247), and the like). Examples of suitable vectors include HIV-1, HIV-2, HIV-3, HIV-14, HIV-15, HIV-16, HIV-17, HIV-20, HIV-21, HIV-22, HIV-23, HIV-24, HIV-19, HIV-25, HIV-26, HIV-30, HIV-40, HIV-41, HIV-42, HIV-43, HIV-44, HIV-45, HIV-53, HIV-54, HIV-60, HIV-70, HIV-80, HIV-11, HIV-124, HIV-13, HIV-14, HIV-15, HIV-16, HIV-17, HIV-18, HIV-29, HIV-20, HIV-21, HIV-21, HIV-22, HIV-24, HIV-25, HIV-30, HIV-31, HIV-32, HIV-33, HIV-34, HIV-35, HIV-45, HIV-46, HIV-47, HIV-52, HIV-53, HIV-54, HIV-65, HIV-70, HIV-81, HIV-14, HIV-15, HIV-16, HIV-17, HIV-18, HIV-19, HIV-20, HIV-21, HIV-22, HIV-24, HIV-25, HIV-35, HIV-19, HIV-21, HIV-22, HIV-34, HIV-19, HIV-25, HIV-26, HIV-27, HIV-35, HIV-36, HIV-37, HIV-40, HIV-41, HIV-42, HIV-53, HIV-

[0111] Non-viral delivery vehicles and methods can also be used, including, but not limited to, polycation-condensed DNA (see, for example, Curiel, Hum. Gene Ther. (1992) 3: 147), with or without linked to killed adenovirus alone, DNA linked to a ligand (see, for example, Wu, J. Biol. Chem. (1989) 264: 16985), eukaryotic cell delivery vehicle cells (see, for example, U.S. Patent No. 5,814,482, PCT Publication No. WO 95 / 07994, PCT Publication No. WO 96 / 17072, PCT Publication No. WO 95 / 30763, and PCT Publication No. WO 97 / 42338), and nuclear charge neutralization or fusion with cell membranes. Naked DNA can also be used. Exemplary naked DNA transfer methods are described in PCT Publication No. WO 90 / 11092 and U.S. Patent No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Patent No. 5,422,120, PCT Publication No. WO 95 / 13796, PCT Publication No. WO 94 / 23697, PCT Publication No. WO 91 / 14445, and European Patent No. 0524968. Additional techniques are described in Philip, Mol. Cell. Biol. (1994) 14:2411 and Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

[0112] The specific dosing regimen used in the methods described herein, i.e., dosage, timing, and repetition, will depend on the particular subject and the subject's medical history. In some embodiments, multiple antibodies, or a combination of an antibody and another appropriate therapeutic agent, may be administered to a subject in need of treatment. The antibody may also be used in conjunction with other agents that enhance and / or complement the effectiveness of the agent. The effectiveness of treatment for the target disease / disorder can be evaluated by methods well known in the art.

[0113] Anti-sADAM9v2 antibodies and treatment methods involving those described in this disclosure and others can be used in combination with other types of treatment for the target disease or disorder disclosed herein. The term "in combination" in this context means that the antibody composition and the therapeutic agent are given either simultaneously or sequentially. Examples include chemotherapy, immunotherapy (e.g., treatment involving anti-inflammatory drugs, immunosuppressants, therapeutic antibodies, antibodies, CAR T cells, or cancer vaccines), surgery, radiation, gene therapy, etc., or anti-infective therapy. Such treatments can be administered simultaneously or sequentially (in any order) with the treatments according to the present disclosure.

[0114] When the antibody compositions described herein are used concomitantly with a second therapeutic agent, subtherapeutic dosages of either the composition or the second agent, or both, can be used in treating a subject having or at risk of developing a disease or disorder associated with sADAM9v2-mediated cell signaling. As used herein, a "subtherapeutic dose" refers to a dosage that is less than the dosage that produces a therapeutic result in a subject when administered in the absence of another agent or agents. Thus, a subtherapeutic dose of a drug is one that does not produce the desired therapeutic result in a subject in the absence of administration of an anti-sADAM9v2 antibody described herein. Therapeutic dosages for many clinically used drugs are well known in the medical field, and further therapeutic dosages can be determined by one of skill in the art without undue experimentation. Therapeutic dosages are extensively described in references, such as Remington's Pharmaceutical Sciences, 18th Edition, 1990, and numerous other medical references relied upon by medical professionals for guidance in the treatment of diseases and disorders. For additional useful agents, see also: Physician's Desk Reference, 59th ed. (2005), Thomson PDR, Montvale NJ; Gennaro et al., eds., Remington's The Science and Practice of Pharmacy, 20th ed. (2000), Lippincott Williams and Wilkins, Baltimore, MD; Braunwald et al., eds., Harrison's Principles of Internal Medicine, 15th ed. (2001), McGraw Hill, NY; Berkow et al., eds., The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway, NJ.

[0115] V. Diagnostic Applications Any of the anti-sADAM9v2 antibodies disclosed herein can also be used to detect the presence of sADAM9v2 (e.g., secreted sADAM9v2) in vitro or in vivo. Results obtained from such detection methods can be used for diagnostic purposes (e.g., diagnosing diseases associated with secreted sADAM9v2) or for scientific research purposes (e.g., identifying new sADAM9v2-secreting cell types, studying the biological activity and / or regulation of secreted sADAM9v2). For assay uses, e.g., diagnostic uses, the anti-sADAM9v2 antibodies described herein can be conjugated to a detectable label (e.g., an imaging agent, e.g., a contrast agent) to detect the presence of sADAM9v2 (e.g., secreted sADAM9v2) either in vivo or in vitro. As used herein, "conjugated" or "attached" means that two entities are associated, preferably with sufficient affinity, that a therapeutic / diagnostic benefit of the association between the two entities is realized. The association between the two entities can be either direct or via a linker, such as a polymeric linker.

[0116] Conjugates or attachments can include covalent or non-covalent bonds, as well as other forms of association, such as entrapment of one entity on or within the other, or entrapment of one or both entities on or within a third entity, such as a micelle.

[0117] In one example, the anti-sADAM9v2 antibodies described herein can be attached to a detectable label, which is a compound capable of emitting a detectable signal, either directly or indirectly, so that the aptamer can be detected, measured, and / or characterized in vitro or in vivo. Examples of such "detectable labels" are intended to include, but are not limited to, affinity tags, e.g., fluorescent labels, chemiluminescent labels, colorimetric labels, enzyme markers, radioisotopes, and biotin. Such labels can be conjugated to the aptamer directly or indirectly by conventional methods.

[0118] In some embodiments, the detectable label is an agent suitable for detecting sADAM9v2-secreting cells in vitro, which can be a radioactive molecule, a radiopharmaceutical, or an iron oxide particle. Suitable radioactive molecules for in vivo imaging include, but are not limited to: 122 I, 123 I, 124 I, 125 I, 131 I, 18 F, 75 Br, 76 Br, 77 Br, 211 At, 225 Ac, 177 Lu, 153 Sm, 186 Re, 188 Re, 67 Cu, 213 Bi, 212 Bi, 212 Pb, and 67 Exemplary radiopharmaceuticals suitable for in vivo imaging include: 111 In oxyquinoline, 131 I sodium iodide, 99 mTc mebrofenin, and 99 mTc red blood cells, 123 I sodium iodide, 99 mTc Examethadime, 99 mTc macroaggregated albumin, 99 mTc medronate,99 mTc mertiatide, 99 mTc oxidronate, 99 mTc pentetate, 99 mTc pertechnetate, 99 mTc sestamibi, 99 mTc sulfur colloid, 99 Examples include mTc tetrofosmin, thallium-201, or xenon-133.

[0119] The reporter agent can also be a dye, eg, a fluorophore, useful in detecting diseases mediated by sADAM9v2-secreting cells in tissue samples.

[0120] To perform the diagnostic assay in vitro, an anti-sADAM9v2 antibody can be contacted with a sample suspected of containing sADAM9v2, such as sADAM9v2-secreting cells in a disease microenvironment or soluble sADAM9v2. The antibody and the sample can be incubated under appropriate conditions for an appropriate period of time to allow binding of the antibody to the sADAM9v2 antigen. Such interaction can then be detected by routine methods, such as ELISA, histological staining, or FACS.

[0121] To perform a diagnostic assay in vivo, an appropriate amount of an anti-sADAM9v2 antibody conjugated to a label (e.g., an imaging or contrast agent) can be administered to a subject in need of testing. The presence of the labeled antibody can be detected by routine methods based on the signal emitted by the label.

[0122] To conduct scientific research assays, anti-sADAM9v2 antibodies can be used to study the biological activity of sADAM9v2, detect the presence of sADAM9v2 intracellularly, and / or modulate the effects of secreted sADAM9v2. For example, an appropriate amount of anti-sADAM9v2 can be contacted with a sample suspected of producing sADAM9v2 (e.g., a new cell type not previously identified as an sADAM9v2-producing cell). The cells are permeabilized before contacting with the anti-sADAM9v2 antibody. The antibody and sample can be incubated under appropriate conditions for an appropriate period of time to allow binding of the antibody to the sADAM9v2 antigen. Such interaction can then be detected by routine methods, such as ELISA, histological staining, or FACS.

[0123] VI. Kits for Therapeutic and Diagnostic Applications The present disclosure also provides kits for the therapeutic or diagnostic applications disclosed herein. Such kits can include one or more containers containing an anti-sADAM9v2 antibody, such as any of those described herein.

[0124] In some embodiments, the kit can include instructions for use according to any of the methods described herein. The included instructions can include instructions for administering an anti-sADAM9v2 antibody to treat, delay the onset of, or alleviate a target disease described herein. The kit can further include instructions for selecting an appropriate individual for treatment based on identifying whether the individual has the target disease. In yet other embodiments, the instructions include instructions for administering the antibody to an individual at risk for the target disease.

[0125] Instructions for use of anti-sADAM9v2 antibodies generally include information regarding the dosage, dosing schedule, and route of administration for the intended treatment. Containers may be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. Instructions provided in kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0126] The label or package insert indicates that the composition is used for treating, delaying the onset of, and / or alleviating a disease or disorder treatable by modulating the immune response, such as an autoimmune disease. Instructions can be provided for practicing any of the methods described herein.

[0127] The kits of the present invention are in suitable packaging, including but not limited to vials, bottles, jars, soft packaging (e.g., sealed Mylar or plastic bags), and the like.

[0128] Also contemplated are packages for use in combination with certain devices, such as inhalers, nasal administration devices (e.g., atomizers), or infusion devices, such as minipumps. The kits may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an anti-sADAM9v2 antibody described herein.

[0129] The kit may optionally provide additional components, such as buffers and interpretive information. Typically, the kit includes a container and a label or package insert on or associated with the container. In some embodiments, the invention provides an article of manufacture comprising the contents of the kit described above.

[0130] Also provided herein are kits for use in detecting secreted sADAM9v2 in a sample. Such kits may include any of the anti-sADAM9v2 antibodies described herein. In some examples, the anti-sADAM9v2 antibody can be conjugated to a detectable label described herein. As used herein, "conjugated" or "attached" means that two entities are associated, preferably with sufficient affinity, such that the therapeutic / diagnostic benefit of the association between the two entities is realized. The association between the two entities can be either direct or via a linker, e.g., a polymer linker. Conjugation or attachment can include covalent or non-covalent bonding, as well as other forms of association, such as entrapment of one entity on or within the other, or entrapment of one or both entities on or within a third entity, e.g., a micelle.

[0131] Alternatively, or in addition, the kit may include a secondary antibody capable of binding to the anti-sADAM9v2 antibody. The kit may further include instructions for using the anti-sADAM9v2 antibody to detect secreted sADAM9v2.

[0132] VII. General Techniques The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989) Cold Spring Harbor Press, Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press, Animal Cell Culture (RI Freshney, ed., 1987), Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-98) J. Wiley and Sons, Methods in Enzymology (Academic Press, Inc.), Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.), Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987), Current Protocols in Molecular Biology (eds. F.M. Ausubel et al., 1987), PCR: The Polymerase Chain Reaction, (eds. Mullis et al., 1994), Current Protocols in Immunology (eds. J.E. Coligan et al., 1991), Short Protocols in Molecular Biology (Wiley & Sons, 1999), Immunobiology (C.A. Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: a practical approach (D. Catty, ed., IRL Press, 1988-1989), Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, ed., Oxford University Press, 2000), Using antibodies: a laboratory manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and J.D. Capra, ed., Harwood Academic Publishers, 1995). Without further elaboration, it is believed that one skilled in the art can, based on the preceding description, utilize the present invention to its fullest extent. The following specific embodiments, therefore, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purpose or subject matter discussed herein.

[0133] Without further elaboration, it is believed that one skilled in the art can, based on the preceding description, utilize the present invention to its fullest extent. The following specific embodiments, therefore, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purpose or subject matter discussed therein.

[0134] Discovery of sADAM9v2 In PCR analysis evaluating ADAM9 expression, discrepancies were observed in the length of some ADAM9 sequences compared to a previously determined benchmark (see Figure 1, indicated by red arrows). This study sought to confirm the expression of the ADAM9 gene in various prostate cancer cell lines and control samples. These included NC (a negative control representing the absence of prostate cancer samples), PC (representing the ADAM9 gene as a positive control), BPH-1 (benign prostatic hyperplasia-1), LNCaP (a prostate cancer cell line derived from a patient with lymph node metastasis), C4-2 (an androgen-insensitive derivative of LNCaP), C4-2B (an indicator of bone metastatic propensity of C4-2), CWR22Rv1 (a prostate cancer cell line representing an androgen mutation), DU145 (a prostate cancer cell line derived from a patient with brain metastasis), PC3 (a prostate cancer cell line derived from a patient with bone metastasis), and PC3M (a cell derivative of PC3 that exhibits pronounced metastatic behavior in a mouse model). The primary objective of this study was to confirm the expression of ADAM9 mRNA in the aforementioned prostate cancer cell lines. RNA was isolated using the Qiagen RNA isolation system (supplied by Qiagen, Inc.) and subsequently converted to cDNA via the MMLV reverse transcriptase system. The PCR procedure was facilitated by incorporating the cDNA into PCR reagents (provided by ThermoFisher, Inc.) and performing PCR analysis via agarose gel electrophoresis. The findings revealed that these cell lines possessed not only the complete ADAM9 mRNA sequence but also various variants. The results highlight the potential for ADAM9 messenger RNA to exhibit distinct alternative splicing repeats in prostate cancer cells, which may contribute to the malignant and metastatic progression of prostate cancer. Therefore, we isolated these PCR bands and performed gene sequencing.

[0135] This is an illustration of the ADAM9 gene after full gene sequencing, where we found that alternative splicing variants exist (see Figure 2). Note that ADAM9-splicing form 1 exhibits alternative splicing between position 2046 in exon 18 and position 2147 in exon 19. This results in out-of-frame splicing and premature termination of ADAM9 translation. As a result, there is loss of the transmembrane and cytoplasmic domains of full-length ADAM9. Furthermore, we identified a novel alternative splicing event demonstrating a loss between position 2040 in exon 18 and position 2236 in exon 19. This results in an omission in the transmembrane domain, but the cytoplasmic sequence remains in frame.

[0136] Further examination of culture medium from tumors, tumor-associated pericytes, and normal tissues revealed that sADAM9 was exclusively detected in the medium from tumors and tumor-associated pericytes (see Figure 3). Cells were cultured for a 3-day period before collection, and then proteins in the medium were concentrated. Concurrently, cultured cells were harvested and protein extraction was performed using a protein extraction system (RIPA buffer containing 150 mM NaCl, 1% Triton-X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris-HCl, pH 8.0). This study aimed to examine the expression of the ADAM9 gene across various prostate cancer cell lines and corresponding control samples. The set included NC (a control showing the absence of prostate cancer samples), PC (serving as a positive control for the ADAM9 gene), BPH-1 (benign prostatic hyperplasia-1), LNCaP (a prostate cancer cell line derived from a patient with lymph node metastasis), C4-2 (an androgen-insensitive derivative of LNCaP), C4-2B (a prostate cancer cell line with a bone metastasis tendency of C4-2), CWR22Rv1 (a prostate cancer cell line expressing an androgen mutation), DU145 (a prostate cancer cell line derived from a patient with brain metastasis), PC3 (a prostate cancer cell line derived from a patient demonstrating bone metastasis), and PC3M (a cell line derived from PC3 that shows a significant metastatic tendency in a mouse model). For protein analysis, standard Western blotting procedures were performed using 8% polyacrylamide gels. Proteins were transferred from the gel to a nitrocellulose membrane via electrophoresis. A specific antibody specifically directed against ADAM9 (MAB939) from R&D Biosystems was used for Western blotting analysis.

[0137] In a specific embodiment, an ELISA system is used to perform a blood test to evaluate the expression of sADAM9 in patients with benign prostatic hyperplasia (BPH) and prostate cancer. As illustrated in Figure 4, the expression of sADAM9 in prostate cancer patients is significantly higher than that in BPH patients. Serum samples from BPH and prostate cancer patients, including 10 samples each from BPH and prostate cancer patients, were obtained from the Taipei Medical University (TMU) Biobank with anonymized identities. ELISA analysis was performed according to the standard protocol provided by R&D Biosystems, Inc., and the corresponding ELISA kit (DY939) was obtained from the same company.

[0138] As depicted in Figure 5, sADAM9v2 expression in cancer-associated periphery cells was determined using laser capture microdissection (LCM) from three patients, exceeding that in benign counterparts from the same patients. To confirm that sADAM9v2 is primarily expressed in prostate tumor-associated periphery cells, LCM was performed on tissue samples from three different patients. These tissue slides, provided by the TMU Biobank, were pathologist-validated for tumor and periphery regions. The LCM procedure followed the guidelines of a commercially available kit (Arcturus® Kit for DNA and RNA Extraction and Purification, Invitrogen, Inc.). LCM operations were facilitated using the MMI CellCut and Cell Ector system available at the TMU Core Facility. RNA from these cells was subsequently isolated using the Arcturus® PicoPure® Frozen RNA Isolation Kit, and cDNA was synthesized using the MMLV cDNA system (Thermo Fisher Scientific, Inc.). Quantitative PCR analysis was performed on a LightCycler® 480 system (Roche Diagnostics, Inc.). Fold differences were calculated using the 2^delta-delta CT analysis method as outlined by the NIH (Biostat. Bioinforma Biomath. Published August 2013, 3(3): 71-85). Final results were expressed in units of fold change derived by comparing cancer vs. benign outcomes.

[0139] These studies confirm that sADAM9v2 is expressed in tumor cells and pericytes. The present disclosure performed structural simulations of ADAM9v2 and found that the alternative splice site, as indicated by the arrow, is highly antigenic (see Figure 6).

[0140] Hereafter, this disclosure will examine the specificity of sADAM9v2 against hybridomas. Cell lysates and conditioned medium (CM) of sADAM9v1 and sADAM9v2 were also collected to test the specificity of the antibodies. The results are shown in Figure 7. We selected the mouse antibodies from which ADAM9 mAbs were generated for the following tests. The NMRI mouse strain was used to generate sADAM9v2 hybridomas and mAbs. All animal procedures followed recommended national and international standards for animal care and handling. The first step involved preparing the immunoconjugate according to a conventional coupling procedure using N-(-3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC). The peptide and its carrier (bovine serum albumin) were combined in equal proportions and suspended in phosphate-buffered saline (PBS), supplemented with 50 mM NaHCO3 buffer. We selected 6- to 12-week-old NMRI mice for immunization and administered 150 micrograms of immunoconjugate in 200 microliters of PBS combined with 100 microliters of complete Freund's adjuvant. A booster shot featuring the same amount of immunoconjugate was administered six weeks later. Blood samples were collected seven weeks after the initial injection. Murine SP2 / 0 cells were used for hybridoma development. These cells were cultured in a solution containing 10 mL of RPMI-1640, 10% FBS, and 2 mM L-glutamine (5 mL). Seven weeks after immunization, the mice were euthanized, and spleen cell suspensions were prepared. These were then suspended in a balanced salt buffer (BSS) consisting of 125 mM NaCl, 5 mM KCl, 4 mM ClCl2, 2.5 mM MgCl2, and 5 mM Tris-HCl, pH 7.4. The mixture was then combined with PEG8000 to balance the splenocyte-to-myeloma cell ratio, facilitating electroporation, and then suspended in HAT medium for one week. After allowing the cultures to stabilize, hybridoma cloning was performed. We ultimately identified a monoclonal hybridoma that was confirmed to produce a mAb with significant specificity against ADAM9v2.

[0141] Preparation of Recombinant Human sADAM9v mAb: After identifying distinct hybridomas suitable for generating sADAM9v mAb, we performed isotype evaluation and typing to determine the unique isotype of the sADAM9v mAb present in the hybridomas. Isotype screening was facilitated using a hybridoma isotyping ELISA system provided by Thermo Fisher Scientific. After validating the isotype, sequencing analysis was performed for the specific antibody isotypes present in the hybridomas. After confirming distinct sequences for both the heavy and light chains, we proceeded to isolate and clone the specific sequences associated with sADAM9v mAb. This was achieved by PCR targeting the designated sequences, which were then incorporated into TGEX-heavy chain and TGEX-light chain expression vectors for subsequent application. The TGEX vectors harboring the heavy and light chains of sADAM9v mAb ensure consistent and conservative expression of our claimed antibody within our system.

[0142] sADAM9-Induced Prostate Cancer Cell Migration: To evaluate the behavior of prostate cancer cells after induction of sADAM9, we used a Transwell cell migration assay using the Corning® system. We obtained recombinant ADAM9 (rADAM9) from R&D Biosystems® for incorporation into the transwell setup. The observed data revealed that sADAM9 (in the form of rADAM9) promoted prostate cancer cell migration in a dose-proportional manner (shown in the left panel). Furthermore, the inclusion of a mAb against sADAM9v2 inhibited cell migration activity within the transwell system, suggesting that the sADAM9v mAb functions as a neutralizing antibody (nAb).

[0143] The results are shown in Figure 8, which shows that the migration of prostate cancer cells is dose-dependent on the concentration of sADAM9, and the generated sADAM9v mAb can reduce the migration of prostate cancer cells.

[0144] Our previous findings highlighted the specificity of sADAM9v mAb and its role in reducing the motility of prostate cancer cells. It has been well documented that the migratory and invasive behavior of prostate cancer cells is linked to downstream cellular regulation of AKT and Src. Consequently, we conducted research on the AKT (referred to in Int. J. Mol. Sci., June 2020; 21(12): 4507) and Src pathways (referred to in Cancer and Metastasis Reviews, February 2014; 33: 595-606). Therefore, following the above studies, the present disclosure further attempts to discover the mechanism of sADAM9 in increasing prostate cancer cell movement and migration, and to discover that sADAM9 activates downstream effectors of AKT to increase prostate cancer cell movement and migration. Protein assays revealed elevated levels of AKT phosphorylation. Prostate cancer cells were grown on either collagen-1 (accounting for the abundance of collagen in bone)-coated or uncoated plates. Prior to harvesting, cells were treated with 5 μg / ml sADAM9 for 30 or 60 minutes. Cell lysis was performed using protein lysis buffer (RIPA buffer) supplemented with phosphatase inhibitors. AKT and Src activity was validated by assessing their phosphorylation levels using antibodies specifically targeting the phosphorylated forms of AKT and Src obtained from Cell Signaling®.

[0145] As shown in Figure 9, sADAM9 has the ability to enhance downstream signals of cell motility, such as AKT phosphorylation. Furthermore, as shown in Figure 8, sADAM9v mAb blocks cell motility. Therefore, we hypothesize that sADAM9v mAb blocks prostate cancer motility through inhibition of AKT phosphorylation.

[0146] Other embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only one example of a generic series of equivalent or similar features.

[0147] From the above description, those skilled in the art can easily ascertain the essential features of the present invention, and can make various changes and modifications to the present invention to adapt it to various usages and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are within the scope of the appended claims.

[0148] equivalent While several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or obtaining one or more of the results and / or advantages described herein. Each such variation and / or modification is deemed to be within the scope of the embodiments of the present invention described herein. More generally, those skilled in the art will readily recognize that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the specific application or applications for which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the present invention described herein. Accordingly, it is to be understood that the foregoing embodiments are presented by way of example only, and that, within the scope of the appended claims and their equivalents, embodiments of the present invention may be practiced other than as specifically described and claimed. The inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the inventive scope of the present disclosure, where such features, systems, articles, materials, kits, and / or methods are not inconsistent with one another.

[0149] All definitions and uses herein should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or the conventional meaning of the defined term.

[0150] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is mentioned, which may include the entire document in some cases.

[0151] The indefinite articles "a" and "an," as used herein and in the claims, unless expressly indicated otherwise, should be understood to mean "at least one."

[0152] The term "and / or," as used herein and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are present conjunctively in some cases and disjunctively in other cases. Multiple elements listed with "and / or," i.e., "one or more" of the elements so conjoined, should be construed in the same manner. Other elements, whether related or unrelated to the elements specifically identified by the "and / or" clause, may optionally be present. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language, such as "comprising," can, in one embodiment, refer to A only (optionally including elements other than B); in another embodiment, refer to B only (optionally including elements other than A); in yet another embodiment, refer to both A and B (optionally including other elements); and so forth.

[0153] As used herein and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., the inclusion of at least one, but also including more than one of a list of possible elements, and optionally additional unlisted items. Only terms clearly indicated otherwise, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," refer to the inclusion of exactly one element of a list of possible elements. In general, the term "or" as used herein should be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") only when preceded by exclusive terms, such as "either," "one of," "only one of," or "exactly one of." When used in the claims, "consisting essentially of" has its ordinary meaning as used in the field of patent law.

[0154] The phrase "at least one," as used herein and in the claims, in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, including, but not necessarily, at least one of every single element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to the specifically identified element. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") can refer in one embodiment to at least one A, optionally including more than one, and no B (and optionally including elements other than B); in another embodiment to at least one B, optionally including more than one, and no A (and optionally including elements other than A); in yet another embodiment to at least one A, optionally including more than one, and at least one B, optionally including more than one (and optionally including other elements); etc.

[0155] It will also be understood that, unless expressly stated otherwise, in any method claimed in the present invention including multiple steps or actions, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are recited. The above amino acid sequence list is shown in Table 1 as follows: [Table 1] [Accession number]

[0156] ATCC VR-67 ATCC VR-1247 ATCC VR-373 ATCC VR-1246 ATCC VR-923 ATCC VR-1250 ATCC VR 1249 ATCC VR-532

Claims

1. An antibody or antigen-binding fragment thereof that binds to sADAM9v2 protein, wherein the antibody or antigen-binding fragment thereof is CDR1 comprising the amino acid sequence of SEQ ID NO: 1; CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 4; CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR3 comprising the amino acid sequence of SEQ ID NO: 6 Light chain containing An antibody or antigen-binding fragment thereof comprising:

2. a heavy chain variable region comprising an amino acid sequence having at least 90% identity to SEQ ID NO:7; and a light chain variable region comprising an amino acid sequence having at least 90% identity to SEQ ID NO:8 The antibody or antigen-binding fragment thereof of claim 1, comprising:

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:

9.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the light chain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:

10.

5. The antibody or antigen-binding fragment thereof of claim 1, conjugated to a therapeutic agent, a fluorescent label, a chemiluminescent label, a colorimetric label, an enzymatic marker, a radioisotope, and an affinity tag.

6. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

7. A reagent for predicting or diagnosing a sADAM9v2-associated disease, for determining the effectiveness of a drug after treatment with a sADAM9v2 inhibitor, or for screening subjects for whom treatment with a sADAM9v2 inhibitor will be highly effective, the reagent comprising an antibody or its antigen-binding fragment described in any one of claims 1 to 5.

8. A composition for a method for predicting or diagnosing an sADAM9v2-associated disease or a predisposition to developing said sADAM9v2-associated disease in a subject, comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 5, said method comprising the steps of: (a) contacting a sample isolated from said subject with said composition; (b) detecting sADAM9v2 protein in the sample by detecting binding between the antibody or antigen-binding fragment thereof and the sample; and (c) comparing the level of the sADAM9v2 protein in the sample with a control, wherein a higher level of sADAM9v2 protein than the control indicates that the subject is suffering from or at risk of developing the disease. A composition comprising:

9. The composition of claim 8, wherein the sADAM9v2-associated disease is a cancer that expresses sADAM9v2.

10. The composition of claim 9, wherein the cancer is prostate cancer.

11. Use of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5 for the manufacture of a pharmaceutical composition for treating an sADAM9v2-associated disease.

12. A pharmaceutical composition comprising an effective dose of the antibody or antigen-binding fragment thereof of any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

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